Synthesis and in vitro Translation of Luciferase Reporters

pT7-FLuc-A50, pT7-PV-FLuc-A50, and pT7-EMCV-FLuc-A50 were linearized with NotI and transcribed with HiScribe T7 High yield RNA Synthesis Kit according to manufacturer's instructions (New England Biolabs). For NanoLuc reporters with differing 5′ UTR lengths, pNL1.1 (Promega) was used as a PCR template. Forward primers contained a T7 promoter for transcription and were positioned to generate the indicated 5′ UTR length. Reverse primer added a synthetic poly(A) tail. RNAs was transcribed with HiScribe T7 High yield RNA Synthesis Kit according to manufacturer's instructions (New England Biolabs). In vitro translation assays were performed as previously described (4 (link)).

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Lyons S.M., Kharel P., Akiyama Y., Ojha S., Dave D., Tsvetkov V., Merrick W., Ivanov P, & Anderson P. (2020). eIF4G has intrinsic G-quadruplex binding activity that is required for tiRNA function. Nucleic Acids Research, 48(11), 6223-6233.

Publication 2020

HiScribe T7 High yield RNA Synthesis Kit

Assays Emcv Nanoluc Poly a tail Primer Promega Synthesis Transcription

Corresponding Organization : Harvard University

Other organizations : Tohoku University, Sechenov University, Federal Medical-Biological Agency, A.V. Topchiev Institute of Petrochemical Synthesis, Russian Academy of Sciences, Case Western Reserve University

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Variable analysis

independent variables

Promoter type (pT7-FLuc-A50, pT7-PV-FLuc-A50, pT7-EMCV-FLuc-A50)
5' UTR length (for NanoLuc reporters)

dependent variables

Transcription and translation efficiency (as measured by in vitro translation assays)

control variables

Linearization of plasmids with NotI
Transcription with HiScribe T7 High yield RNA Synthesis Kit (New England Biolabs)
In vitro translation assay procedure (as previously described)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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